Reproduction (2002) 124, 733743

Review

Current status of sexing mammalian spermatozoa

George E. Seidel, Jr1 and Duane L. Garner2
1Animal Reproduction and Biotechnology Laboratory, Colorado State University,

Fort Collins, CO 80523, USA; and 2XY, Inc., Fort Collins, CO 80523, USA

Thousands of offspring have now been produced via artificial insemination with
spermatozoa sexed by flow cytometry and cell sorting. We are unaware of any other
practical approach to sexing spermatozoa that maintains fertility. Accuracy of sexing
usually is 8595% in most species, but somewhat lower with human spermatozoa.
Spermatozoa are sexed in series, one at a time, at routine rates of about 3000 live
spermatozoa of each sex per second for most species, and nearly twice that rate under
optimal conditions for some species. Owing to various constraints and statistical
considerations, there appears to be an upper theoretical limit to sexing spermatozoa of
about 10 000 live spermatozoa of each sex per second with current methodology. About a
quarter of the spermatozoa processed are sexed; the rest are discarded in the process or
lost due to logistical constraints. Spermatozoa undergo some damage during sorting,
although much less in terms of viability than with routine cryopreservation; fertility is
lower with sexed than control spermatozoa. Offspring from sexed spermatozoa appear to
have no more abnormalities than do controls, and both groups grow and thrive similarly.
Despite high costs and complex procedures, sexing spermatozoa, usually followed by
cryopreservation, is being used commercially for cattle and horse production in several
countries, and is used to produce girls to avoid X-chromosome-linked genetic diseases.

Introduction
It is now possible to predetermine the sex of offspring from
a number of species before fertilization with an accuracy
of 8595% (Seidel et al., 1999; Welch and Johnson,
1999; Johnson, 2000). This noteworthy accomplishment,
first demonstrated convincingly by Johnson et al. (1989),
resulted from integration of advances in many fields
including chromosomal karyotyping, artificial insemination, maintenance of spermatozoa fertility in vitro, DNAspecific staining, flow cytometry, computer science and
high speed cell sorting. Advances from these different
disciplines were integrated by a body of innovative scientists co-operating from several sites including Lawrence
Livermore National Laboratory, the United States Department
of Agriculture Beltsville Agricultural Research Center,
Cambridge University, Colorado State University, Atlantic
Breeders Cooperative and the company, XY, Inc. (Johnson
and Seidel, 1999; Garner, 2001).
The objectives of this review are to explain: (1) why
spermatozoa bearing the X- or Y-chromosome are so similar
phenotypically, but what is different between them; (2) the
principles and procedures used to sex spermatozoa by total
DNA content via flow cytometry and cell sorting; (3) the
accuracy, speed, and efficiency of current sperm sexing
Email: gseidel@colostate.edu

procedures, particularly explaining why the majority of

spermatozoa used are not included in the final sorted
product; (4) the extent to which spermatozoa are damaged
during current sexing procedures, including effects on
fertility, and normality of calves resulting from sexed
spermatozoa; and (5) methods other than measuring DNA
content, of sexing spermatozoa under recent or current
investigation.

Spermatozoa differ in sex chromosome size but in

little else
Historical perspective
The first documented microscopic identification of sex
chromosomes was by Guyer (1910). This observation, along
with those of others, generated the idea that mammalian
sex might be controlled by these specialized chromatin
structures. A variety of techniques, mostly unsuccessful,
were used in an attempt to identify which sex chromosome
was contained in an individual spermatozoon, with the
ultimate goal of separating these gametes. The first convincingly documented difference between mammalian
X- and Y-chromosome-bearing spermatozoa was the differential uptake of quinacrine stain by human sex chromosomes (Barlow and Vosa, 1970). The heterochromatin of
the Y-chromosome fluoresced more brightly than other
chromosomes, including the X-chromosome; however, this

2002 Society for Reproduction and Fertility

1470-1626/2002

G. E. Seidel, Jr and D. L. Garner

734

After centromeres divide, these will form

two X-chromosome-bearing spermatozoa

After centromeres divide, these will form

two Y-chromosome-bearing spermatozoa

Fig. 1. Karyogram of chromosomes that would be present in two adjacent bovine secondary
spermatocytes derived from a primary spermatocyte. The total length of chromosomes of bovine
X-chromosome-bearing spermatozoa is about 3.8% greater than that of Y-chromosome-bearing
spermatozoa.

difference did not occur in chromosomes and spermatozoa

from non-primates.

the range 34.5% (Johnson et al., 1987; Johnson, 2000) but

in a few species the difference is much larger.

50:50 Sex ratios

DNA-binding dyes

Sex is determined in mammals by whether the fertilizing

spermatozoon contains an X-chromosome to produce a
female or a Y-chromosome to produce a male. As a consequence of the way that chromosomes segregate at meiosis,
the chance that a spermatozoon will carry either chromosome is equal. Nature has gone to extremes to minimize
phenotypic differences (for example, size, shape, surface
properties) between spermatozoa carrying different alleles
and different sex chromosomes (Seidel, 1999). Phenotypic
equivalence of mammalian spermatozoa within males,
despite major allelic differences, is ensured by at least four
mechanisms: (1) heterochromatic sex chromosomes are
encased in sex vesicles post-meiotically; (2) intercytoplasmic bridges between spermatocytes and spermatids
allow interchange of molecules including mRNA; (3) there
is limited post-meiotic expression of most autosomal genes
during the later stages of spermiogenesis, due in part to
extreme condensation of chromatin; and (4) spermatozoa
are coated by high-affinity proteinaceous secretions originating from Sertoli cells, excurrent ducts and accessory sex
glands, that render the surface of spermatozoa anonymous
with regard to possible sex-specific and other allelic differences of the cell membrane (Seidel, 1999).

The strong binding of certain fluorescent dyes to nucleic

acids enables precise quantification of sperm nuclear DNA,
in some cases without affecting sperm viability. Many
dyes have been used, but only after application of the
bisbenzimidazole Hoechst 33342 (2-(4-ethoxyphenyl)-5(4-methyl-1-piperazinyl)-2,5-bi-1H-benzimidazole3 HCl)
for staining intact spermatozoa was fluorescence quantification of the DNA content of living spermatozoa successful
(Johnson et al., 1987). Hoechst 33342 (H33342) is a livecell stain that permeates the cell membrane and binds
selectively to AT base pairs along the minor groove of
dsDNA. It is a yellow solid having a formula weight of 561.9
(C27H28N6O3HCl) and is moderately water-soluble and
relatively non-toxic. H33342 usually is excited with the 351
or 364 nm lines of an argon-ion laser or other sources of
fluorescence excitation such as mercury lamps and exhibits
a relatively large Stokes shift (excitation/emission maxima of
about 350/460 nm), making it very useful in assessing
precise amounts of DNA in cells.
Most DNA stains intercalate between the base pairs of
the DNA, thereby presumably increasing their mutagenicity. However, H33342 is not an intercalative dye, which
probably makes it safer to use (Watkins et al., 1996). Nevertheless, H33342 may be toxic to workers at high doses, so it
must be used carefully. Binding of H33342 to DNA is
stabilized by a combination of hydrogen bonding, van der
Waals forces, and electrostatic interactions between the dye
molecule and the negatively charged DNA molecule. The
strength of this multiple-binding interaction may contribute
to the putative radioprotective properties of the H33342
molecule (Young and Hill, 1989).
Dead or moribund cells in the population of spermatozoa stained with H33342 can be identified by adding
propidium iodide (Johnson et al., 1994). More recently,
this classical dead-cell stain has been replaced with red

Karyograms
Karyograms are displays of chromosomes at metaphase
and are used to assess normalcy of numbers and shapes
of chromosomes characteristic of a particular species, and
to identify sex. For example, a normal male diploid bovine
karyogram consists of 58 autosomes plus an X and a Y
chromosome (all duplicated). A bovine karyogram is
illustrated (Fig. 1). The difference in DNA content between
X- and Y-chromosome-bearing bovine spermatozoa is
approximately 3.8%; differences for most mammals are in

Sexing mammalian spermatozoa

(a) Overview of sperm sorter; computer not shown.

735

(b) Optics and hydrodynamics

Cytonozzle with
XY orient-tip
Side fluorescence
objective:
cell orientation
Blocking bar
Forward
fluorescence
objective:
cell DNA content
Exiting stream

Pressure
control

Sorting
area

Laser MLUV
333363 nm

Sample Sample
port introduction

(c) Within the Cytonozzle is a piezo

crystal. Frequency waves are
applied to the crystal, which is
coupled to the fluid inside the
nozzle. This causes the stream to
break off into droplets at controlled
intervals. Sperm sorters typically
produce 70 000 drops per second
with a 70 m diameter nozzle.
The sperm sorter is calibrated so
that a spermatozoon of the desired
sex is inside the last attached drop
when a positive (for Y-chromosomebearing spermatozoa) or negative
(for X-chromosome-bearing
spermatozoa) charge is applied to
the stream. The drop that carries
that spermatozoon breaks away
from the stream holding the charge.
The drops then travel between high
voltage fields which direct them to
their respective collection tubes.
Drops in this figure are about
240 m in diameter.
Last attached drop

This figure shows the objectives for the forward and side
fluorescence detectors, Cytonozzle with orienting tip,
and blocking bars to keep the incidental light from the
beam from reaching the detectors. Forward fluorescence
measures DNA content and side fluorescence measures
cell orientation of spermatozoa in the exiting stream.

3500 V

+3500 V

(d) Both X- and Y-chromosome-bearing spermatozoa

can be sorted simultaneously.
Droplets containing
Y-chromosome-bearing
spermatozoa are given a
positive charge, so are
attracted to the negative field
to the left whereas droplets
containing X-chromosomebearing spermatozoa are
given a negative charge and
are attracted to the right.
The respective streams of
droplets are collected in
separate test tubes that
contain an egg yolk medium
to keep spermatozoa
healthy.

Y-chromo- Waste X-chromosome- stream somebearing

bearing
spermaspermatozoa
tozoa

Droplets with no spermatozoa, dead spermatozoa, or

unsexable spermatozoa are
uncharged, and drop straight
into the waste stream.

Fig. 2. MoFlo SX spermatozoa sorter.

food dye (FD&C40) to avoid potential mutagenic effects

of propidium iodide (Johnson and Welch, 1999; Schenk
et al., 1999). Other food dyes also are effective. The
mechanism of action is to quench the H33342 fluorescence
of spermatozoa that have damaged membranes so that they
can be removed during the sorting process by dead-cell
gating.

Separation of spermatozoa by DNA content via flow

cytometry and cell sorting
Detailed procedures for flow-sorting mammalian spermatozoa are beyond the scope of this review, and depend on
the species and application. However, an overview will be
provided.

G. E. Seidel, Jr and D. L. Garner

736

Box 1. Steps in sexing spermatozoa

The following steps summarize the overall process for
sexing bull spermatozoa. More details are given by Schenk
et al. (1999).
(1) Collect semen.
(2) Store up to 9 h undiluted at 2023C until staining.
(3) Dilute an aliquot in staining medium to 200 106
spermatozoa ml1.
(4) Stain aliquot with H33342 for 45 min at 34C.
(5) Dilute in sorting medium to 100 106 spermatozoa
ml1; add food colouring.
(6) Sort batch for 90 min.
(7) Repeat steps 36 every 90 min for up to 9 h from the
same semen sample.
(8) Concentrate sample to approximately 40 106 spermatozoa ml1 by centrifugation at 850 g for 20 min
and discard the supernatant. Sorted spermatozoa are
diluted (to approximately 8 105 spermatozoa ml1)
as a consequence of mixing sorting medium, catch
fluid and sheath fluid (Fig. 2d).
(9) Accumulate several sort batches and pool.

Steps applied to semen during sorting

The process used by XY, Inc. for sorting bull spermatozoa
(see Box 1) will be used as a framework for this section. For
nearly all applications, freshly collected semen is used.
Spermatozoa that have been cryopreserved and thawed
do not tolerate the sexing process well, particularly if
they are to be re-frozen. Since sperm sorting is a slow
process relative to the needs for most applications, keeping
spermatozoa healthy between semen collection and sorting
is important. Schenk et al. (1999) have done several studies
on this step with bull semen, and of the many procedures
tried, storing the semen undiluted at 2023C for 08 h was
the best procedure. Storage of bovine spermatozoa for
longer than 8 h has not been studied thoroughly in the
context of flow-sorting, although limited studies with
various methods have shown that dilution during storage
has adverse effects on H33342 DNA-staining properties.
On the other hand, stallion spermatozoa are sexed routinely
after storage in appropriate diluents at 515C for 18 h,
resulting in excellent fertility if spermatozoa are not
cryopreserved after sorting. Logistics dictate the staining of a
new batch of bovine spermatozoa from the raw semen
every 12 h, thus minimizing the time of exposure of
spermatozoa to high concentrations of dye, and minimizing
the destaining that occurs during dilution between staining
and sorting.
In subsequent steps of the sorting process (Box 1),
spermatozoa reside in a number of fluids: staining medium;
sorting medium; a mixture of catch fluid, sheath fluid and
sorting medium; and fluids for preparation of the final
product after concentration by centrifugation, for example,
cryoprotectant media. The medium for staining spermato-

zoa with H33342 is a modified Tyrodes albumin lactate

pyruvate (TALP) (Schenk et al., 1999). This is diluted with
TALP containing egg yolk to which vital-staining food dye is
added for sorting. During the sorting process, spermatozoa
in the sorting medium are mixed with Tris-based sheath
fluid containing citric acid and fructose. Sheath fluid
functions as a cylindrical wall of fluid surrounding the core
stream of sorting medium (containing the spermatozoa) that
guides the flow of fluid through the flow cytometer. The
exiting fluid is composed of approximately 90% sheath fluid
and 10% core stream fluid. After spermatozoa pass through
the sorter, the streams of X- and Y-chromosome-bearing
spermatozoa are collected into tubes containing a 22% egg
yolk-Tris extender (catch fluid; Schenk et al., 1999). As the
test tube fills during sorting (Fig. 2d), the initial catch fluid is
continually diluted by the stream exiting the sorter nozzle.
The sorting, sheath and catch fluids obviously need to
be compatible with maintaining sperm fertility, but respective fluids also must have certain electrical and viscosity
properties, as well as not interfere with staining and
fluorescence.
Spermatozoa that accumulate every 34 h are frozen as
a batch, so there are usually two freezing batches for
each ejaculate, each freezing batch consisting of several
sorting batches. In many instances, two or three sorters
(up to ten at one location) are used simultaneously for
the same ejaculate to improve the scale of operation. This
procedure enables freezing of reasonable numbers of doses
of spermatozoa per batch every 34 h, and minimizes the
time between sorting and cryopreservation of spermatozoa.

Principles of flow cytometer and cell sorter use for sexing

spermatozoa
Components of a flow cytometer and cell sorter designed
for sexing spermatozoa are illustrated (Fig. 2). In brief, as a
spermatozoon passes two fluorescence detectors at 90
angles to each other (Fig. 2b), each detector measures the
intensity of fluorescence resulting from excitation of
the DNA-bound dye molecules by light, which usually is
generated by a laser. The wavelengths of light used depend
on the light source, and if and how the light is filtered.
Importantly, if, for example, an argon laser is used as a light
source, spermatozoa are not exposed to the damaging low
ultraviolet wavelengths that are absorbed by nucleic acids
and proteins.
The strength of the fluorescence signals obviously
depends on the number of fluorescing molecules bound to
DNA. This is the basis for sexing spermatozoa. In addition,
the signal depends on a number of other parameters,
including the laser intensity (Guthrie et al., 2002), whether
and how the laser is pulsed, optical properties of the entire
system, sensitivity of detectors and electronic noise. All of
these factors must be kept as constant as possible to resolve
the small differences in DNA content between X- and
Y-chromosome-bearing spermatozoa.
The main obstacle to accurate quantification of sper-

Sexing mammalian spermatozoa

matozoa DNA with this approach is the geometry of the

sperm head, which is paddle-shaped in most species of
interest. The intensity of fluorescence is lowest if the
flat face of the paddle is oriented toward a detector, and
highest when the edge is so oriented. Flat orientation results
in the most accurate discrimination between X- and Ychromosome-bearing spermatozoa, so only spermatozoa
oriented in this way are sorted (Fig. 2b). The second
detector at 90 to the laser is used to diagnose orientation
(Fig. 2b). As the fluorescence signal is highest for spermatozoa oriented with their paddle edge toward this 90
detector, only the population of spermatozoa that emit peak
fluorescence to the 90 detector are considered oriented
appropriately for sexing by the contemporaneous signal to
the 0 detector (Fig. 2b).
Since sperm orientation will be random with respect to
detectors in a cylindrical stream of fluid, only about 10% of
spermatozoa will be sufficiently well oriented for accurate
DNA measurements under those circumstances. A considerable effort, therefore, has been made to increase this percentage by modifying the cylindrical geometry of the fluid
stream (Johnson and Welch, 1999; Rens et al., 1998, 1999).
Although details will not be reviewed here, about 70% of
spermatozoa are oriented correctly with current technology
(Johnson and Welch, 1999) (Fig. 2b). Other approaches
to this problem, such as measuring total fluorescence
with spherical detectors (Sharpe et al., 1997) have not yet
become useful.

How fast can high-speed flow cytometers and cell sorters

sex spermatozoa?
Modern flow cytometers sort spermatozoa at rates that
are commercially viable by propelling spermatozoa through
the system at accelerating speeds approaching 90 km h1
when they exit the nozzle. Discrete fluorescence signals
from two detectors at 90 angles to each other are produced
at a rate of over 180 000 measurements per detector per
second, and the information is processed by computer
and relayed to the stream-charging mechanism by the time
that the spermatozoa have travelled a few cm, such that
the droplets to be formed with X-chromosome-bearing
spermatozoa have different electrical charges from those
with Y-chromosome-bearing spermatozoa. Computation is
so fast and sensitive that should a droplet contain two X- or
Y-chromosome-bearing spermatozoa by chance, even these
droplets can be saved, while droplets with dead or misoriented spermatozoa can be discarded.
Sorting of nearly all non-human spermatozoa for sex
currently is done with the high pressure, high speed, multipurpose flow cytometer built by Cytomation, Inc. (Fort
Collins, CO), model MoFlo SX equipped with an argon
laser, detectors for fluorescence, and a special spermatozoa
orientation nozzle modified from one developed by Rens
et al. (1998, 1999). Detailed technical specifications are
beyond the scope of this review, but a key parameter is flow
rate past the detectors, which is over 20 m s1. Theoretically,

737

as spermatozoa are about 100 m in length, if they were

perfectly oriented in a column head-to-tail, about 200 000
spermatozoa could pass the detector each second, and
even more if the size of sperm heads only were considered.
Unfortunately, the distribution of spermatozoa in such a
column approximates a Poisson distribution. This means
that there is considerable space between each flowing
spermatozoon. If the concentration of spermatozoa is
increased, while this average space shortens, more and
more spermatozoa are relatively clumped, so that for practical purposes, fluorescence signals between them are too
close together for the system to process them separately,
resulting in more and more unusable coincidence signals.
A second, related problem is that some spermatozoa are
statistically between droplets, so it is unclear in which
droplet (Fig. 2c) the spermatozoa will reside; in this case,
both drops are discarded (termed an abort). Theoretically
and empirically, the optimum throughput in this statistical
situation is achieved at about 25 000 spermatozoa per
second, the so-called event rate. Event rates can be changed
by modifying the concentration of spermatozoa in the
sample, either directly or by changing the ratio of the core
stream to sheath fluid. However, there is no net benefit
above about 25 000 spermatozoa per second due to
increasing rates of abortion or coincidence. Another option
is to increase the flow rate past the detector by increasing
the pressure of the entire system. However, this similarly
results in increased rates of abortion or coincidence and
damage to the spermatozoa.
Superimposed on the above considerations are physical
constraints of droplet size. Droplets are formed as the
column of fluid exits the nozzle due to vibrations set up by
a piezoelectric mechanism in the column, even before the
spermatozoa pass the detectors. The frequency of vibrations
must be matched with characteristics of the nozzle tip,
particularly diameter, as well as viscosity of the fluid and
system pressure. Roughly, with a nozzle with an internal
diameter of 70 m, nearly 70 000 droplets can be formed
per second at pressures of 40 psi, but fewer at lower
pressures and larger orifice diameters. The ideal situation is
to make as many droplets per unit time as possible without
disturbing other properties of the system such as efficacy of
sperm orientation.
Matching a Poisson distribution of 25 000 spermatozoa
per second in the column of fluid to discrete droplets of
a specific size produced at 70 000 per second occurs
imperfectly. About 25% of droplets will contain a single
spermatozoon, a few per cent will contain two or more
spermatozoa, and > 70% will not contain any spermatozoa.
There is no simple way around these inefficiencies as they
are inherent due to laws of statistics and physics. They place
an upper limit on sorter performance at approximately
80 000 droplets per second at 50 psi and sorting about
10 000 live spermatozoa of each sex per second per nozzle,
assuming perfect sperm orientation and perfect resolution of
oriented spermatozoa. A better option may be lower
pressures and fewer droplets, resulting in less sperm

G. E. Seidel, Jr and D. L. Garner

738

Box 2. Typical efficiency of sexing spermatozoa

Aliquot of stained spermatozoa (100%)
(1) Residual loss of spermatozoa in staining tube
(2) Losses in sorter tubing between batches,
between males and to prevent or correct
plugged nozzles

10%

12%

Spermatozoa that are evaluated (78%)

(3) Spermatozoa discarded due to malorientation 30%
(4) Spermatozoa discarded due to coincidence 15%
(5) Discarded dead spermatozoa
10%
Potentially sortable spermatozoa (35%)
(6) Spermatozoa discarded to maintain purity 12+%
(Fig. 3) because distributions of X- and
Y-chromosome-bearing spermatozoa
fluorescence overlap
(7) Probable aneuploid spermatozoa discarded
1%
(Fig. 3)
(8) Spermatozoa discarded due to aborts and
2%
droplets with both X- and Y-chromosomebearing spermatozoa
Spermatozoa that have been sorted (30%)
(9) Spermatozoa lost because of spraying (missing 4%
the fluid in the bottom of the collection tube
Fig. 2d)
(10) Losses of spermatozoa in the supernatant
15%
after centrifugation (concentration step)
(11) Loss of spermatozoa during filling and sealing 4%
straws including incomplete volume of residue
in the last straw
Sorted spermatozoa that are frozen (23%)
(12) Use of spermatozoa for quality control
(accuracy and motility after thawing)
Spermatozoa available for insemination
(22%; 11% of each sex at 90% accuracy)

4%

Percentage values in the right column refer to percentages of the

respective sub-headings rather than of the starting material.

damage but slower sorting speeds. To circumvent these

limits would require fundamental changes in sorting
procedures, changes that seem unlikely to be developed
over the next few years.

Efficiency of sperm sorting

There is potential for sperm losses at virtually every
step of processing and sorting (see Box 2). The losses are
highly dependent on the staining properties of a particular
ejaculate as well as on the skill and the care taken by
the technicians doing the work. Losses are multiplicative.
Typical cumulative efficiency for the 12 sequential steps in
Box 2 at 90% accuracy of sorting is 22% (11% of each sex).
Of course, 10% of the spermatozoa were dead and were
purposely discarded.

The above efficiencies vary as a result of the quality of

the sample and the speed of sperm flow through the system.
Sometimes ejaculates degrade during the day and the efficiencies listed become much lower, particularly at step 1,
but also at steps 5 and 6. About 5% of the time, the entire
days work is discarded because the motility of spermatozoa
after thawing does not meet quality control standards, as
also occurs with unsexed spermatozoa. As fewer sexed
spermatozoa usually are packaged per insemination dose
than conventionally, these losses do not necessarily lead to
fewer inseminates per volume of semen used than occur
with normal semen processing with standard numbers of
spermatozoa.
Under most practical circumstances, only part of an
ejaculate will be sorted because the process is slow relative
to the number of spermatozoa available. As spermatozoa
will undergo various stresses during the sorting process,
fresh samples seem to tolerate sorting significantly better
than gametes that have been stored for hours. The above
considerations, plus the number of sperm sorters available
will determine how many spermatozoa in an ejaculate
should be held for sorting, and how many will be available
for non-sorted applications.
The raw data from flow cytometers can be displayed
in many ways. The simplest way is to present only the
fluorescence data from the 0 detector for adequately
oriented, live spermatozoa (Fig. 3), that is those showing
maximal fluorescence with the 90 detector (see Johnson
and Welch, 1999). Misoriented and dead spermatozoa
are not considered in Fig. 3; droplets containing them and
spermatozoa not resolvable due to coincidence would
receive no electric charge when exiting the nozzle of the
sorter and, thus, would be discarded in the waste stream
(Fig. 2d). Thus, the fluorescence from the 0 detector of
the oriented, live subset of resolvable spermatozoa (less
than half of the spermatozoa monitored) can be plotted as
in Fig. 3. Note that the distributions of fluorescence of
the X- and Y-chromosome-bearing spermatozoa overlap,
resulting in an overall bimodal distribution. By discarding
the spermatozoa in the centre of the bimodal distribution
(shaded in Fig. 3), the spermatozoa to the left are primarily
Y-chromosome-bearing spermatozoa and those to the right
are primarily X-chromosome-bearing spermatozoa. The
width of the shaded area in Fig. 3 generally is set to produce
spermatozoa sexed at 90% accuracy. Spermatozoa in the
extreme tails of the curve often have missing (left tail) or
extra chromosomes (right tail); these aneuploid spermatozoa also are discarded by not charging droplets containing them.

Damage to spermatozoa and normality of calves

Damage to spermatozoa during sorting
Fertility of sorted spermatozoa is somewhat lower than
that of controls (Seidel et al., 1999; Buchanan et al., 2000)
as is survival of sorted spermatozoa after cryopreservation

Number (frequency) of live, oriented spermatozoa

Sexing mammalian spermatozoa

739

of two curves
(what is actually observed)

X spermatozoa

Y spermatozoa

Aneuploid, e.g.
monosomic
spermatozoa

94

Contaminating
X spermatozoa

96

Contaminating
Y spermatozoa

98
100
102
Normalized fluorescence units

Aneuploid, e.g.
trisomic
spermatozoa

104

106

Fig. 3. Theoretical histograms illustrating sorting efficiencies of X- and Y-chromosome-bearing bovine

spermatozoa recovered from the sorter. The initial sample would contain 50% Y- (blue) and 50%
X-chromosome-bearing (pink) spermatozoa. The shaded area between the X- and Y-chromosome-bearing
spermatozoa peaks show magnitude of the overlap, where it is impossible to distinguish between
spermatozoa carrying an X-chromosome and those with a Y-chromosome. These spermatozoa are
discarded. The wider the shaded area, the purer the X- and Y-chromosome-bearing spermatozoa
populations will be, but the more spermatozoa that will be discarded.

(Schenk et al., 1999). Unfortunately, most studies on fertility

with sexed spermatozoa are confounded by using fewer
sexed spermatozoa per insemination dose than normal
procedures would dictate. When similar numbers of
spermatozoa per dose have been used, pregnancy rates
with sexed spermatozoa usually have been 6080% of
unsexed control spermatozoa (Doyle et al., 1999; Seidel
et al., 1999). Pregnancy losses in cattle between 1 and 2
months of gestation have been 12 % higher with low
insemination doses of sexed spermatozoa than with normal
insemination doses of unsexed spermatozoa (Seidel et al.,
1999). It will take large numbers of animals to determine
whether this is a true effect, or whether this non-significant
difference will dissipate with more thorough study.
One recent study concerns the amount of damage to
DNA and mortality of spermatozoa subjected to various
combinations of mechanical forces at 50 psi, exposure to
laser, and staining procedures during sorting (Fig. 4; Garner
et al., 2001). Sorted spermatozoa were further analysed by
flow cytometry for both DNA integrity (Evenson, 1989) and

failure to exclude propidium iodide as a measure of dead

spermatozoa. It is clear (Fig. 4) that most of the damage
resulted simply from processing the spermatozoa through
the sorter, even with no staining and no exposure to laser
light. Additional damage due to exposure to laser and dye
was small and not statistically significant, in agreement with
other studies (Libbus et al., 1987; Guthrie et al., 2002). Very
recent studies from our laboratory (Suh and Schenk, in
press) indicate that much of the mechanical damage noted
can be alleviated by lowering the pressure of the fluid
during sorting. This is likely to improve fertility of sexed
spermatozoa compared with studies to date in which spermatozoa have been sorted at 50 psi.

Normalcy of offspring of sex-sorted spermatozoa

Several thousand offspring from seven mammalian
species (cattle, pigs, rabbits, horses, sheep, elk and humans),
primarily cattle, have been produced after H33342-staining
and flow-sorting of the fertilizing spermatozoa. No gross

Percentage of COMPT

10

100

80

60

40

20
0

0
Unsorted

Sorted

Laser

Ho33342

Percentage of dead spermatozoa

G. E. Seidel, Jr and D. L. Garner

740

Laser/
Ho33342

Fig. 4. Percentages of () dead spermatozoa and () spermatozoa with damaged DNA after thawing
as determined by the spermatozoa chromatin stability assay (percentage of COMPT, cells outside of
the main population) after (1) unsorted control, (2) passing spermatozoa through the sorter without
laser or staining, (3) with laser but no staining, (4) with staining, but no laser, and (5) with both staining
and laser (modified from Garner et al., 2001).

abnormalities have been reported (Morrell and Dresser,

1989; Johnson, 1995; Catt et al., 1997; Doyle et al., 1999;
Fugger, 1999; Johnson and Welch, 1999; Seidel et al.,
1999), although we are aware of an anecdotal report of one
abnormal calf. These data indicate that the DNA of flowsorted spermatozoa that result in offspring is not severely
damaged. Here, we report preliminary results from a large,
ongoing study.
Black Angus heifers were inseminated with frozen, sexed
and unsexed control spermatozoa during 3 days. Standard
sexing procedures were used (Schenk et al., 1999; Seidel
et al., 1999). Inseminations were balanced over semen
from two bulls, four inseminators, two doses of sexed spermatozoa (1.5 106 and 4.5 106 frozen spermatozoa per
insemination dose) and a control (20 106 frozen, unsexed
spermatozoa), and two insemination times (12 and 24 h
after oestrus). However, all inseminations were carried out
blind with respect to treatments. Some heifers were inseminated with X-chromosome-bearing spermatozoa, and others
with Y-chromosome-bearing spermatozoa, depending on
pedigrees and perceived commercial value of the resulting
calves. Detailed fertility results will be reported elsewhere;
briefly, there was no significant difference in 2 month
pregnancy rates determined by ultrasonography between
the two doses of sexed spermatozoa (overall average, 53%;
n = 245), which, however, were lower (P < 0.05) than
control pregnancy rates (66%; n = 126).
The pregnant heifers from this study were then allocated
to three different farms and managed by routine husbandry
procedures, but with different intensity of management.
Personnel managing the cattle were unaware of which
heifers were pregnant with sexed or control spermatozoa.
Between 2 months of pregnancy and term, there were four

abortions of 86 (4.7%) control pregnancies and seven

abortions of 130 (5.4%) pregnancies from sexed spermatozoa, a very small, non-significant difference.
There were no significant differences in rates of neonatal
death or accumulated deaths to weaning between calves
derived from sorted versus control spermatozoa, nor were
there any treatment effects on duration of gestation, birth
weight or weaning weight (Table 1). No congenital abnormalities were observed. The only significant effects
(P < 0.01) were that male calves were heavier at birth and
weaning than female calves. Therefore, there appear to be
no detrimental effects of sorting spermatozoa on resulting
offspring. There were large differences in the survival of
calves among the three farms, with deaths to weaning
ranging from 5 to 18%. Such routinely observed differences
in husbandry from farm to farm obviously are much greater
than any effects of sexing spermatozoa. Although this data
set resulting from 371 inseminations has a respectable
sample size (123 calves from sorted sperm; 82 from
controls), more extensive data need to be examined to
be certain that there is no small increase in problems of
offspring resulting from sorted spermatozoa. In addition, the
fate of the minute quantity of H33342 in the female
reproductive tract from the spermatozoa that do not fertilize
oocytes probably merits further study.

Methods for sexing spermatozoa other than DNA

quantification with DNA binding dyes
In addition to fluorescence flow cytometry and cell sorting
based on DNA content, separation of spermatozoa containing the X-chromosome from those with the Y-chromosome
has been attempted with a variety of other techniques

Sexing mammalian spermatozoa

741

Table 1. Characteristics of calves produced from frozen flow-sorted and control spermatozoa

Treatment
Sorteda
Sorteda
Control
Control

Sex of calf
at birth
Male
Female
Male
Female

Mean duration
of gestation
(days) SE

Number of
calves that died
neonatally

Mean
birth weight
(kg) SEb

59
64
48
34

281.0 0.72
280.3 0.65
280.1 0.61
280.8 0.70

2 (3.4%)
6 (9.4%)
4 (8.3%)
3 (8.8%)

33.5 0.72
30.6 0.63
34.4 0.74
31.2 0.87

Number of
calves that died
up to weaning
3 (5.1%)
8 (12.5%)
8 (16.7%)
3 (8.8%)

Mean weaning
weight (kg) SEb
267 3.4
259 3.8
267 4.8
261 5.9

aAccuracy of sorting was 56 of 59 males (95%) produced with Y-sorted spermatozoa, and 62 of 64 females (97%) produced with X-sorted spermatozoa.
bThe only significant differences among treatments were that male calves were heavier than female calves (P < 0.01) at birth and weaning.

(Amann and Seidel, 1982). A brief update of recent and

ongoing work with these technologies follows.

Gradient swim-down procedure

Successful separation of X- and Y-chromosome-bearing
human spermatozoa using an albumin gradient was first
reported by Ericsson et al. (1973). The conceptual basis for
this method is that Y-chromosome-bearing spermatozoa are
smaller in size and exhibit a greater downward swimming
velocity than X-chromosome-bearing spermatozoa within
vertical columns of high density human serum albumin
(Ericsson et al., 1973). A fraction enriched with Ychromosome-bearing spermatozoa can be obtained by
harvesting the first 22% of spermatozoa to swim to the
bottom of the gradient, and discarding the remainder
(Ericsson and Ericsson, 1999). Ericsson and Ericsson (1999)
reported that the latest version of this technique increased
the percentage of male children born to 7080%. However,
the validity of sex pre-selection by this approach has been
challenged repeatedly (for example, Evans et al., 1975).
This technique has never been shown to sex spermatozoa
accurately from mammals other than humans (Beal et al.,
1984; White et al., 1984). Furthermore, it is not possible
ethically to do prospective, randomized, blind trials with
sexed human spermatozoa rigorously to document the true
efficacy of this technique. Another interesting aspect of the
use of this method is that when women are treated with
clomiphene citrate to induce ovulation before insemination, the sex ratio is reversed, so that up to 73% females are
born (Ericsson and Ericsson, 1999).

Surface antigenic differences

Various immunological approaches to sexing spermatozoa of mammals have been tested without repeatable
success (Hoppe and Koo, 1984; Hendriksen et al., 1996;
Hendriksen, 1999). One approach was to target H-Y antigen.
However this molecule appears to be on both X- and
Y-chromosome-bearing spermatozoa (Hoppe and Koo, 1984)
and is possibly derived from Sertoli cells. An immunological
approach, however, would be highly desirable because
inexpensive batch processing could be used to enrich for
either X- or Y-chromosome-bearing spermatozoa. Howes

et al. (1997) suggested that their inability to detect sex-specific

differences in spermatozoa surface antigenicity using rigorous biochemical methods indicates that an immunological
approach to semen sexing was unlikely to work. Nonetheless, attempts using this approach recur regularly.
A recent report suggests that a viable immunological
sperm sexing procedure can be developed using a more
rigorous method to isolate sex-specific proteins (SSPs)
(Blecher et al., 1999). In this newer approach, non-SSPs
were removed immunologically before the attempted isolation of SSPs because they are likely to be more highly conserved than non-SSPs. Antibodies to SSPs were raised and
used to identify SSPs by affinity chromatography (Blecher
et al., 1999). Antibodies to purified female fetal SSPs caused
agglutination of approximately half of the bovine spermatozoa and when the unagglutinated spermatozoa were
isolated and used in bovine IVF, they produced > 90%
male embryos (Blecher et al., 1999). This immunological
approach, which implies post-meiotic transcription or translation of SSPs that do not equilibrate through inter-spermatid
cytoplasmic bridges, appeared promising. However, to date
there are no reports of producing sex-selected offspring with
this procedure despite considerable investment of resources
over several years.

Free-flow electrophoresis
Electrophoretic separation of mammalian sex determining spermatozoa has been attempted by many investigators without significant success (Kiddy and Hafs, 1971;
Mohri et al., 1987). This approach, which is based on
the possibility that the electric charge on the surface of
X-chromosome-bearing spermatozoa differs from that exhibited by Y-chromosome-bearing spermatozoa, uses an electric field to separate spermatozoa into the two major classes
(Kaneko et al., 1984). Spermatozoa are introduced into the
free-flow apparatus continuously and move along different
paths to be collected as two main fractions. F-body examination of separated human spermatozoa using quinacrine
staining indicated that the purported X-chromosomebearing fraction was relatively pure, but that the spermatozoa in the Y-chromosome-bearing fraction were not (Kaneko
et al., 1984). However, the resulting spermatozoa were
compromised because sperm motility was reduced

742

G. E. Seidel, Jr and D. L. Garner

significantly. Successful separation of sex-determining

spermatozoa of other mammalian species has not been
reported, although this approach may currently be undergoing reappraisal.

Sperm sorting based on volumetric differences

Spermatozoa containing an X chromosome are theoretically larger than those containing a Y chromosome.
van Munster et al. (1999a) recently used interference
microscopy and subsequent image analysis to demonstrate
a difference in sperm head volume that matched differences
in DNA content between X- and Y-chromosome-bearing
bovine spermatozoa. A method based on this principle has
been developed for sorting live spermatozoa by using
interference microscopy optics with a flow cytometer (van
Munster, 2002). Such a method, which eliminates the need
to use DNA-specific dyes, would be a highly attractive
alternative method for sexing mammalian spermatozoa.
Unfortunately, the potential purity of spermatozoa separated using volumetric measurements cannot exceed 80%
purity of either sex based on theoretical considerations (van
Munster et al., 1999b), and recent efforts to make this
practical have not been encouraging (van Munster, 2002).

Centrifugal countercurrent distribution

Recently, Ollero et al. (2000) have attempted to sex ram
spermatozoa by centrifugal countercurrent distribution
using an aqueous two-phase system. This is a chromatographic process that partitions cells into a stationary, lower
phase and a mobile, upper phase, repeated numerous
times. Centrifugation was used to speed the partitioning
process, so a set of 59 partitions was done in about 1 h.
Ollero et al. (2000) found that they could obtain fractions
of up to 75% Y-chromosome-bearing spermatozoa with
reasonable viability using this procedure at certain salt concentrations. However, they presented no data on repeatability of the process or fertility of the spermatozoa. Each
batch produced about 6 106 spermatozoa (75% Ychromosome-bearing). This procedure needs to be verified;
it also may not be as successful for species with less difference in DNA content between X- and Y-chromosomebearing spermatozoa than in sheep (4.2%; Johnson, 1995).

Genetic approaches
Some years ago, a genetic approach to sperm sexing
was suggested (Seidel, 1988), which subsequently has been
demonstrated (Herrmann et al., 1999). This approach
involves the well known transmission distortion ratio of
alleles at the T loci on chromosome 17 in mice. Rather than
having half of the offspring with each of two alleles, > 90%
(the percentage depends on the specific allele and genetic
background) receive the detrimental allele, a violation of
Mendels law of independent assortment. Basically, spermatids with one allele poison those with the other allele,
presumably via intracellular bridges in spermatids. Herrmann

et al. (1999) demonstrated this concept by placing part of

this genetic system on the Y chromosome using transgenic
procedures. They produced a strain of mice that produced
66% males (217/331) in the course of natural mating. In
principle, this could be done in any species, with either sex
chromosome; however, such a project would be expensive
and complicated, and there are some caveats in non-murine
species.

Perspectives on commercialization
Commercialization of sperm sexing has begun for cattle and
is imminent for horses (Buchanan et al., 2000), and the
methodology is being used on a limited scale to produce
human babies, particularly to produce girls to avoid Xlinked genetic disease (Johnson et al., 1993; Fugger et al.,
1998). This review has focused on the actual process of
sexing spermatozoa by flow cytometry and cell sorting, and
illustrates that the procedure is complicated and somewhat
inefficient. However, procedures continue to improve.
Sperm sexing as currently practised is expensive, partly due
to inefficiencies, partly due to personnel costs for the many
steps, and partly due to the high cost of equipment and its
maintenance. Despite these costs and complexities, the
procedure works, and already appears to be commercially
viable for niche applications in several species. Much
simpler equipment designed specifically for sperm sexing
probably will become available within a few years. As
efficiencies improve and costs decline, sperm sexing will be
applied more widely.
The authors gratefully acknowledge the conscientious assistance
of Zell Brink, Michael Evans, John Schenk, Sarah Seidel, Tae-Kwang
Suh, Lisa Tubman, and Sallie Varner in preparing this review.
Reported research was supported financially by XY, Inc., Fort
Collins, CO, USA and the Colorado State University Experiment
Station.

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